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1. (WO1979000880) QUANTITATIVE TESTING FOR VITAMIN B12
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CLAIMS
1. A composition for use in the assay of vitamin B12
(cobalamin) wherein the active component is substantially specific to vitamin B12 and substantially free of substances which react with vitamin B12
analogues.
2. The composition of claim 1 wherein the active com ponent is .a bidding protein.
3. The composition of elaim 2 wherein the binding protei is intrinsic factor.
4. The composition of claim 3 wherein the intrinsic factor is selected from the group consisting of human IF, hog IF and rabbit IF.
5. The composition of claim 4 wherein the intrinsic
factor includes human IF.
6. The composition of claim 4 wherein the intrinsic factor includes hog IF.
7. The composition of claim 1 wherein one or more protein binder which is non-specific to vitamin B12 is present and has been rendered substantially inactive or inoperative as to its ability to bind with vitamin B12 by treatment with a vitamin B12 analogue,
said analogue being substantially, reactive with said non-specific protein and substantially non reactive with the material which is a specific binder for B12, said analogue being present in an amount
sufficient to render substantially all of the nonspecific binder inactive.or inoperative in its
ability to bind with vitamin B12.
8. The composition of claim 7 wherein the specific active binder includes intrinsic factor protein and the non-specific protein binder includes an R protein.

9. The composition of claim 8 wherein a portion of the vitamin B12 analogue is selected from the group consisting of cobinamide, CN-Cbl (bde-OH) and (3,5,6- Me3BZA) (CN,OH) Cba.

10. The composition of claim 7 wherein the vitamin B12 analogue is present in the amount of about a 10-fold to about a 100, 000-fold excess, based of the molar amount of the nort-speciffc protein.
11. The composition of claim 9 wherein the vitamin B12 analogue is present in the amount of about a 1-fold to about a 10,000,000-fold excess based on the molar amount of the nonrspecific protein.
12. The method of preparing ffte binder of claim 2
wherein a composition including binding material
substantially specific to vitamin B12 and binding
materials substantially non-specific to vitamin B12 and including a protein is treated with vitamin B12 analogues to remove substantially all of the non- specific vitamin B12 binding material, said
vitamin B12 anald-gue being substantially reactive
with said non-specific binder and substantially non- reactive with the material which is a specific
binder for B12, said analogue being added in an
amount sufficient to render substantially all of
the non-specific binder inactive or inoperative in its ability to bind with vitamin B12.
13. The method of preparing the binder of claim 7
wherein a composition including protein binding
material substantially specific to vitamin B12 and binding material substantially non-specific to
vitamin B12 is treated with vitamin B12 analogue
to render the binder non-specific to vitamin B12
substantially inactive or inoperative as to its
ability to bind with vitamin B12, said vitamin-B12 analogue being substantially reactive with said
non-specific binder and substantially non-reactive with the material which is a specific binder for
B12, said analogue being added in an amount sufficient to render substantially all of the non-specific binder inactive or inoperative in its ability to
bind with vitamin B12.

τhe -mέthόd of claim- 13 wherein the specific active binder includes intrinsic factor protein and the non - specific . prp te-En-Tbl nd er i nclude s an R prote in.
^Α'fMdioiϋsotope' dilution assay for measuring the vitamin B12 level in a sample comprising:
contactinrg sai d--sample with a knon amount of a radioisotope of Vitamin BiV and a composition corϊ- taining a binding protein substantially specific to vitamin B12, said compositlpn bging substantially Free. of substances which bind Vitamin B12 analogues

The method of claim 15 wheiein the binding protein X is intrinsico factor.
The method ofo claim- 15 wherein the binding Protein x consists essentially of protein selected-rfsbm the group consistιng of human IF hog IF aqd rabbit IF.

The method of claim 15 wherein the soύrce of
vitamin B12 is selected from the group consisting A of mammalian tissue and mammalian blood A

The method of claim 15 whereiruthe source of q,A vitamin B12 is human blood.
The method of measuring vitamin B1 2 anai-o-g-ues present
q , A in a sample which also inciudes Vitamin B12' including the steps of A assaying a pottion of the sample with aMbiDder which is a binder for both vitamin B12 and vitamin B12 analogues;
assaying a portion of the sample with a binder which is sabstantially specific to vitamin B12 and sub- stantially freè of substances which bind vitamin B12 analogues; and
then determining the difference between the two assays, as being substantially indicative of the amount bf vitamin B10 analogue in the sample.